The perfection of plant regeneration capabilities and the application of biotechnological techniques for genome modification provide a highly desirable system for the improvement of crop species.
Investigations concerning the morphogenesis of plant tissue in culture date back at least to the 1950's (Skoog, F. and Miller C. O., Symp Soc. Exp. Biol., 11:118 (1957)) and have continued apace to date. Several monographs provide extensive reviews of the field and contain compilations of numbers of species which will undergo plant regeneration in culture (See for example, Murashige T., In: "Propagation of Higher Plants through Tissue Culture," T. A. Thorpe, Ed., p. 15, Univ. Calgary Press, Calgary (1978); Vasil, I. K. et al. Adv. Gent. 20:127 (1979) and Evans, D. A., et al. In: "Plant Tissue Culture: Methods and Applications in Agriculture: T. A. Thorpe, Ed. pg. 45, Academic Press, N.Y. (1981)).
The impressive list of plants species cited in the above-referenced monographs, for which successful regeneration has been achieved, belies the difficulties in achieving those results. As will be noted later, successful regeneration of a particular species is often characterized by the addition of (or even omission of) catalytic amounts of auxins, cytokinins, or other growth regulators. Further, successful regeneration may also be a function of not only the mere presence of a certain compound but its ratio to other media components as well. Since each plant species appears to possess a relatively unique optimal set of media requirements, the successful preparation and regeneration of a new species cannot be necessarily inferred from the successful regimens applied to unrelated plant varieties.
Despite the recent advances in plant regeneration for a variety of species, corn (Zea mays) is one of the crops which has been refractory to regeneration protocols; hence, the application of plant cell culture for improvement of the majority of pure lines and commercial hybrids of this crop has lagged behind that progress in the field in general.
Absent a functioning regeneration protocol, more traditional avenues for crop improvement have been utilized. One approach has been to introduce into the commercial corn genome agronomically useful characteristics derived from exotic or "wild" Zea germplasm by conventional sexual hybridization and back-crossing breeding procedures. One source of exotic germplasm which has been employed is teosinte (Zea spp.) Teosinte has been described as a possible ancestor of today's modern Zea mays forms. There are several known races of teosinte that are either annual and diploid or perennial and tetraploid. A new teosinte that is both diploid and perennial (Zea diploperennis) has recently been discovered (Iltis, H. H., et al. Science 203: 186-188 (1979)). This species can be sexually hybridized with commercial corn. Interspecies hybrids between Z. mays and Z. diploperennis are fertile and based on nearly complete chromosome pairing will be potentially useful for crop improvement (Pasupulet, C. V. and W. C. Galinat, J. of Heredity 73:168-70 (1982) and Galinat, W. C. and C. V. Pasupulet Maydica 27:213-220 (1982)). Among the more useful traits for which Zea diploperennis may serve as a source include, resistance to maize chlorotic dwarf, maize chlorotic mottle, and maize streak viruses, and maize bushy stunt mycoplasma as well as tolerance to maize raydo fino virus. Unfortunately, even though Zea diploperennis possesses valuable genetic potential for corn improvement, by being limited to conventional breeding techniques, it will require years to develop the improved lines.
It is, therefore, highly desirable to discover conditions which will permit the regeneration of Zea diploperennis from tissue culture thereby allowing the full range of biotechnological techniques to be brought to bear on the breeding process; thereby significantly reducing the time required to recover improved breeding lines.
The culture of the diploid and annual teosinte (Zea mexicana) was undertaken (Cure, W. W. and R. L. Mott Physiol Plant. 42:91-96 (1978)) and limited callus-like growth was reported but no shoot regeneration occurred. It was also reported (Dhaliwal, H. S. and H. Lorz, Maize Genetics Coop. Newsletter 53:144 (1979)) that scutella cultures of immature embryos of F.sub.1 hybrids between teosinte (Z. mexicana) `El Salado` and the inbred corn line `B-73` regenerated numerous plantlets. Shoot culture was also tested with mature seeds of B-73, El Salado and B-73 x El Salado. B-73 corn tissues had no response while El Salado and hybrid tissues gave similar results as those described for immature embryo cultures.
It has not heretofor been possible to employ somatic tissue from field grown plants of Zea diploperennis as a source of material for plant regeneration from tissue culture.
The term "plant tissue culture" as used herein is taken in its broadest meaning to refer to the cultivation, in vitro, of all plant parts, whether a single cell, a tissue or an organ, under aseptic conditions. More restrictive terms relating to plant tissue culture technology include: "callus culture" by which is meant, the culture of cell masses on agar medium and produced from an explant of a seedling or other plant source; "cell culture" by which is meant, the culture of cells in liquid media in vessels which are usually aerated by agitation; "organ culture" by which is meant, the aseptic culture on nutrient media of anthers (microspores), ovaries, roots, shoots, or other plant organs; "meristem culture and morphogenesis" by which is meant, the aseptic culture of shoot meristems or other explant tissue on nutrient media for the purpose of growing complete plants, and "protoplast culture" by which is meant, the aseptic isolation and culture of plant protoplasts from cultured cells or plant tissue.